By means of genetic and biochemical procedures, it is proposed to analyze the mechanisms that control the biosynthesis of the two amino acids, isoleucine and valine, in Escherichia coli. Highest priority will be given to determining the nucleotide sequence of those regions of DNA in the ilv gene cluster that are the sites for transcription initiation and transcription termination, the two processes thought to be the primary means of controlling the levels of the isoleucine and valine biosynthetic enzymes. The source of DNA will be cloned fragments derived from phages carrying defined lengths of the ilv gene cluster. Also important will be the determination of the portions of the ilv gene cluster that are actually transcribed. Another project will be to purify the protein needed for the induction of acetohydroxy acid synthase substrate. Mutations affecting the protein have been found and the effects of these mutations on the binding of inducer to the protein or of the protein to the DNA will be studied. Coupled transcription-translation in vitro with ilv-lac fusion DNA will be employed to define the elements needed for expression of both the "multivalently repressed" ilvEDA operon and the induction of the ilvC gene. The physical and genetic maps of the gene cluster will be further correlated by heteroduplex and restriction enzyme digestion of DNA fragments containing known genetic functions.